Monoclonal antibodies (mAbs) hold great promise in human health with applications ranging from therapeutic agents that target cancer cells, to diagnostic biomarkers that can detect trace levels of a given antigen. This promise is best reflected in global sales of antibodies which reached nearly $31 billion in 2007 and future sales predicted to reach $56 billion by 2012, a compound annual growth rate of 13%. Stoking this rapid growth is recombinant DNA technology, which has led directly to the development of a handful of powerful technologies that are widely exploited to engineer human mAbs and antibody-derived fragments with high affinity and specificity for virtually any target antigen. From a therapeutic standpoint, full-length mAbs or IgGs are often advantageous over smaller antibody fragments due to their long circulating half-life in mammals, which results from a combination of their large molecular size that prevents clearance in the kidneys and their ability to avoid proteolysis in the endothelium by using a salvage pathway. However, due to the complexity of these multi- subunit proteins, their production has largely been restricted to eukaryotic expression systems such as CHO or hybridoma cells and is therefore cumbersome, expensive, time consuming, and not amenable to parallelization. As a result of these shortcomings, the existing technologies for discovery and production of IgGs have struggled to keep pace with the rapidly growing demand for these important biomolecules. To help bridge the technological gap associated with IgG production, Escherichia coli cells represent an attractive option due to their simplicity, rapid growth rate, ease of use and low cost of goods. However, while E. coli has proven to be an excellent host for the expression of smaller antibody fragments such as Fvs, scFvs, Fabs or F(ab')2s, its potential for IgG expression and engineering has not been thoroughly investigated. Therefore, the goal of this proposal is to develop E. coli as a robust vehicle for the discovery, engineering and manufacturing of full-length human IgGs. Under Specific Aim 1, a novel E. coli strain will be engineered that is specifically geared towards high-level expression of recombinant IgGs in the cytoplasmic compartment. Specific Aim 2 of this proposal seeks to develop a unique screening method for direct selection of "cytoclonals" - functional IgGs to a given protein antigen isolated from the cytoplasm of living E. coli cells. This screen will be based on the popular split-protein system widely used for detecting protein-protein interactions. The utility of this screen will be demonstrated by screening a large synthetic library of IgG sequences for cytoclonals against target protein antigens. Unlike most other antibody selection-expression systems, the proposed strategy is a unique integration of assay development, library design, and host cell engineering. Successful completion of these studies will greatly expand the toolkit available for producing and engineering full-length IgGs of different antigen specificities that can be used in basic research, diagnosis and therapy. PUBLIC HEALTH RELEVANCE: Monoclonal antibodies and antibody-based fragments account for >30% of all revenues in the biotechnology market and are used to treat a wide array of human diseases including asthma, autoimmune diseases, bacterial and viral infections, cancer and other diseases. Since antibody therapies are an increasingly large fraction of the drugs in development, with ever escalating increases in the cost of drug development, any improvements to the production or discovery of efficacious antibodies will have a significant impact on human health. Accordingly, this proposal seeks to develop Escherichia coli cells as a technology platform for rapid, low-cost expression and isolation of full-length human monoclonal antibodies against virtually any target protein antigen of interest.